Atrial Natriuretic Peptide (ANP) is a potent natriuretic and vasorelaxant polypeptide which has been isolated from the extracts of mammalian atria. DeBold et al., (1981) Life Sci. 28:89-94; Napier et al., (1984) Ann. Rep. Med. Chem. 19:253-262; Kangawa et al., (1984) Biochem. Biophys. Res. Commun. 118:131-139; Flynn et al., (1983) Biochem. Biophys. Res. Commun. 117:859-865; Napier et al., (1984) Biochem. Biophys. Res. Commun. 120:981-988; Currie et al., (1984) Science 223:67-69; Thibault et al., (1984) FEBS Lett. 167:352-356; Atlas et al., (1984) Nature 309:717-719. These peptides have been given a variety of names (e.g., atriopeptins and cardionatrins), but are now collectively referred to as ANP.
It has been determined from the sequence of cloned cDNA for these peptides that they are all derived from the carboxy-terminal region of a precursor protein whose structure has been recently established. Yamanaka et al., (1984) Nature 309:719-722; Maki et al., (1984) Nature 309:722-724; Oikawa et al., (1984) Nature 309:724-726; Seidman et al., (1984) Science 22:324-326; Flynn et al., (1985) Science 228:323-325. The different sizes of ANP appears to be a result of a difference in post-translational processing or artifactual degradation during isolation. Several synthetic ANPs have also been prepared and shown to contain all the biological properties of the native peptides. Seidah et al., (1984) Proc. Natl. Acad. Sci. USA 81:2640-2644; R. P. Nutt et al., in PEPTIDES 1984 (U. Ragnarsson ed. 1985); Atlas et al., supra.
ANP has been shown to play a significant role in blood-pressure homeostasis, regulation of extracellular fluid volume, and as an antagonist to the hypertensive effects of the renin-angiotensin system and other hormonal and neurotransmitter systems. ANP has been detected in the blood by radioimmunoassay. Gutkowska et al., (1984) Biochem. Biophys. Res. Common. 125:315-323; Tanaka et al., (1984) Biochem. Biophys. Res. Commun. 124:663-668. The biological effects of ANP are mediated through the binding of ANP to specific receptors on cell membranes. The existence of specific receptors has been demonstrated in a variety of kidney, adrenal cortex and vascular tissue. Schenk et al. (1985) J. Biol. Chem. 260:14887-14890; Vandlen et al., (1985) J. Biol. Chem. 260:10889-10892; Misono et al., (1985) Biochem. Biophys. Res. Commun. 130:994-1001; Hirose et al., (1985) Biochem. Biophys. Res. Commun. 130:574-579; Yip et al., (1985) J. Biol. Chem. 260:8229-8232; Schenk et al. (II). (1985) Biochem. Biophys. Res. Commun. 127:433-442; Hirata et al., (1985) Biochem. Biophys. Res. Commun. 128:538-546; Winquist et al., (1984) Proc. Natl. Acad. Sci. USA 81:7661-7664; Napier et al., (1984) Proc. Natl. Acad. Sci. USA 81:5946-5950; Hirata et al., (1984) Biochem. Biophys. Res. Commun. 125:562-568; De Lean et al., Endocrinology 115:1636-1638; De Lean et al., (1984) Life Sci. 35:2311-2318.
Because of the potent biological activity of ANP, regulation of its levels in the blood would be a therapeutic approach to the treatment of such disorders as hypertension, shock, and the like. To establish therapeutic protocols, however, it is necessary to have a sensitive assay for determining the levels of ANP in the blood of mammals. Such an assay could also be used to diagnose ailments such as hypertension. ANP receptor protein, if available, could be readily employed in the these assays. While current native and synthetic ANP, as well as analogs thereof, would allow for the modulation of fluid volume and vascular function by increasing ANP levels, effective therapies may also require ANP levels to be reduced in order to achieve the desired extracellular fluid volume and electrolytic homeostasis. It is possible that soluble fractions of ANP receptor could be used therapeutically to reduce serum levels of ANP.
While various attempts have been made to characterize the ANP receptor, it has not been purified. Furthermore, these attempts at characterization have produced conflicting results. See, e.g., Schenk et al. (I), supra; Vandlen et al., supra; Misono et al., supra; Hirose et al., (1985), supra; Yip et al., supra.
Recent work has suggested that there may be more than one ANP receptor. See Leitman et al. (1986) Biochim. Biophys. Acta 885:74-75; Kuno et al. (1986) J. Biol. Chem. 261:5817-5823 (copurification from rat lung of ANP binding and guanylate cyclase activity). Of additional interest regarding the ANP receptor are Leitman et al. (1986) J. Biol. Chem. 261:11650-11655; Scarborough et al. (1986) J. Biol. Chem. 261:12960-12964; Hayashi et al. (1986) Peptide Chemistry 1985. pp. 27-32; Hirata et al. (1985) Biochem. Biophys. Res. Comm. 132:971-984; Napier et al. (1986) Arch. Biochem. Biophys. 248:516-522.
It would be highly desirable, therefore, if purified ANP receptor protein were available, as well as genes to facilitate its production through recombinant means. Monoclonal antibodies to the receptor protein would also be useful since they could be used to characterize the receptor protein, identify additional tissue expressing receptor protein, and block ANP binding to the receptor.
Several receptor molecules unrelated to the ANP receptor have been isolated and purified in the prior art. Wimalasena et al., (1985) J. Biol. Chem. 260:10689-10697 (porcine LH/hCG receptor); Petruzzelli et al., (1984) Proc. Natl. Acad. Sci. USA 81:3327-3331 (insulin receptor); Schneider et al., (1982) J. Biol. Chem. 257:2664-2673 (LDL receptor).